Bioresource Technology 102 (2011) 47934799

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Bioresource Technology
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Low temperature alkali pretreatment for improving enzymatic digestibility

of sweet sorghum bagasse for ethanol production
Long Wu a, Mitsuhiro Arakane a, Masakazu Ike a, Masahisa Wada b,c, Tomoyuki Takai d, Mitsuru Gau d,
Ken Tokuyasu a,
a
Carbohydrate Laboratory, Food Resource Division, National Food Research Institute, National Agriculture and Food Research Organization (NARO), 2-1-12 Kannondai,
Tsukuba, Ibaraki 305-8642, Japan
b
Department of Biomaterials Science, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan
c
Department of Plant and Environmental New Resources, College of Life Sciences, Kyung Hee University, 1, Seocheon-dong, Giheung-ku, Yongin-si, Gyeonggi-do 446-701,
Republic of Korea
d
Forage Crop Breeding Unit, National Agricultural Research Center for Kyushu Okinawa Region, National Agriculture and Food Research Organization (NARO), 2421 Suya,
Koshi, Kumamoto 861-1192, Japan

a r t i c l e

i n f o

Article history:
Received 14 September 2010
Received in revised form 7 January 2011
Accepted 11 January 2011
Available online 20 January 2011
Keywords:
Lignocellulose
Recalcitrance
Pulping
Delignication
Accessibility

a b s t r a c t
A low temperature alkali pretreatment method was proposed for improving the enzymatic hydrolysis
efciency of lignocellulosic biomass for ethanol production. The effects of the pretreatment on the composition, structure and enzymatic digestibility of sweet sorghum bagasse were investigated. The mechanisms involved in the digestibility improvement were discussed with regard to the major factors
contributing to the biomass recalcitrance. The pretreatment caused slight glucan loss but signicantly
reduced the lignin and xylan contents of the bagasse. Changes in cellulose crystal structure occurred
under certain treatment conditions. The pretreated bagasse exhibited greatly improved enzymatic digestibility, with 24-h glucan saccharication yield reaching as high as 98% using commercially available cellulase and b-glucosidase. The digestibility improvement was largely attributed to the disruption of the
lignin-carbohydrate matrix. The bagasse from a brown midrib (BMR) mutant was more susceptible to
the pretreatment than a non-BMR variety tested, and consequently gave higher efciency of enzymatic
hydrolysis.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
Biofuel, as an alternative renewable energy source, is receiving
increasing attention worldwide because of the imminent depletion
of fossil fuel reserves and the rapid accumulation of atmospheric
greenhouse gases. Presently, the production of the second generation cellulosic ethanol from those abundant and inexpensive nonfood lignocellulosic biomass materials, such as agricultural residues and herbaceous energy crops, has become the focus of research due to its potential advantages (Lynd et al., 1991; Snchez
and Cardona, 2008; Sims et al., 2010).
Lignocellulose consists primarily of plant cell wall materials; it
is a complicated natural composite with three major constituents,
i.e. cellulose, hemicellulose and lignin. The hemicelluloselignin
complex and the crystalline structure of cellulose are largely
responsible for the recalcitrance of lignocellulose to hydrolysis
(Hsu et al., 1980). Presently, the enzymatic degradation of lignocel Corresponding author. Tel.: +81 29 838 7189; fax: +81 29 838 7996.
E-mail address: tokuyasu@affrc.go.jp (K. Tokuyasu).
0960-8524/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.01.023

lulose has generally been considered a feasible way to extract sugars from lignocellulosic biomass for ethanol fermentation. In order
to achieve a high glucose yield for yeast fermentation, much of recent research has focused on the improvement of enzymatic
digestibility of the cellulose through various pretreatments. Currently, thermochemical pretreatment methods for opening up the
structure of lignocellulose have been widely studied (Hendriks
and Zeeman, 2009; Wyman et al., 2005). Pretreatment can selectively remove the structural barriers and increase cellulose accessibility depending on the chemical agents used (e.g. acid, alkali,
oxidant, etc.) and treatment conditions applied. Some thermochemical pretreatment performed at high temperatures
(>150 C), such as dilute sulfuric acid, SO2 steam explosion or
ammonia pretreatment, can improve the enzymatic digestibility
of lignocellulosic biomass to some extent (hgren et al., 2007; Sipos et al., 2009). However, the hydrolysis efciency of cellulose
was still improvable considering the relatively low saccharication
yields and high enzyme loading. Besides, the pretreatment
methods involving high process temperature require higher energy
input and heat-resistant/anti-corrosive pressure cooking equip-

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ments; excessively high temperatures may also aggravate the

degradation of the useful components and the formation of
fermentation inhibitors (Oliva et al., 2006; Panagiotou and Olsson,
2007). On the other hand, the pretreatment methods adopting
moderate temperatures (e.g. lime, ozone, H2O2, etc.) reportedly
had quite inconsistent effect on the enzymatic digestibility of the
biomass (Garcia-Cubero et al., 2009; Kim and Holtzapple, 2006a;
Silverstein et al., 2007). In general, currently available pretreatment techniques can hardly meet the requirements of efcient lignocellulose-to-ethanol conversion.
Pulping is the process of converting lignocellulosic materials to
pulp bers for papermaking. The presently dominant chemical pulping process the kraft process uses a solution of sodium
hydroxide and sodium sulde to dissolve and remove the majority
of lignin that binds cellulose bers together at elevated temperature and pressure to produce pulp consisting of almost pure bers.
The major objective of the pulping process, i.e. to liberate carbohydrate polymers from their complex with lignin, is generally consistent with that of the pretreatment operation focusing on removing
the recalcitrance of lignocellulose for ethanol production. In particular, modern kraft pulp mills are self-sustaining. The spent cooking
liquor is concentrated and combusted to recover the reaction
chemicals and power the mills, which lowers the production cost
and reduces pollution discharge (Japan TAPPI, 2006).
Kim and Holtzapple (2006b) reported that the activation energy of alkali delignication of herbaceous biomass such as corn
stover and sugarcane bagasse was signicantly lower than that
of wood. Preliminary studies also showed that the lignin present
in rice straw, bagasse and some grasses was greatly removed
after the biomass was treated with sodium hydroxide solution
at moderate temperatures for a short period of time, even without the addition of the other pulping chemicals. Accordingly, aiming at overcoming the resistance of lignocellulose to enzymatic
hydrolysis with less energy consumption and improving the efciency of cellulose-to-ethanol conversion, a pretreatment method,
named as low temperature alkali (LTA) pretreatment (in contrast
to the processes operated at temperatures > 100 C), was proposed for treating a target lignocellulosic feedstocksweet sorghum bagasse for cellulosic ethanol production. The proposed
method and the kraft/soda pulping technology shares the same
fundamental principles; therefore, those mature techniques and
equipments used in the pulping process to recover the reaction
chemicals as well as energy are basically applicable to the pretreatment process.
Sweet sorghum (Sorghum bicolor (L.) Moench) is a multi-purpose crop which can be cultivated under a wide range of environmental conditions for simultaneous production of grain, sugar juice
and lignocellulosic bagasse. It is considered one of the promising
herbaceous energy crops because of its high yield in biomass and
fermentable sugars per unit area per unit time and relatively low
input requirements, and has received considerable attention as a
feedstock for bioethanol production (Gnansounou et al., 2005; Bennett and Anex, 2009). The brown midrib (BMR) mutants of sweet
sorghum reportedly had reduced lignin content and showed higher
ber digestibility as silage (Jung and Allen, 1995; Ledgerwood
et al., 2009). This feature may also be benecial to the ethanol production as lignin plays an important part in the biomass recalcitrance. By now, little research on the difference in ethanol
productivity between the BMR and regular sweet sorghum bagasse
has been reported.
The objectives of this study were to investigate the effects of the
proposed pretreatment method on the composition, structure and
enzymatic digestibility of bagasse from two varieties of sweet sorghum (including one BMR mutant), and to clarify the connections
between the compositional/structural changes and the digestibility
of the pretreated bagasse.

2. Methods
2.1. Preparation of bagasse
One kilogram of stems of sweet sorghum cv. Kyushuko4 (BMR
mutant) and cv. SIL05 (both harvested in Kumamoto, Japan in September of 2008, and stored at 20 C until thawed for processing)
were pressed using a bench top juice extractor. The brous solid
residue (bagasse) was washed in hot tap water and pressed again
to remove the residual free sugars. The washing-pressing process
was repeated three times. The bagasse was then dried in a drying
oven at 65 C until constant weigh. The dried bagasse was successively ground using a hammer mill (RD1-15, Grow Engineering, Japan) and a bench top multi blender, and sieved to achieve an
average particle size of less than 0.25 mm. The ground bagasse
was put in air tight zip lock bags and stored in a desiccator at room
temperature before use.
2.2. Pretreatment
2.2.1. Low temperature alkali (LTA) pretreatment
A portion of the ground bagasse (140 mg) was evenly dispersed
into 2.8 ml of sodium hydroxide solution of a certain concentration
in a 15-ml plastic test tube using a tube mixer (alkali to biomass
ratio ranging between 0.1 and 4 g NaOH/g oven dried bagasse).
The mixture was incubated in a shaking water bath at a selected
temperature for a desired length of time, and then centrifuged at
12,000g for 5 min, the supernate discarded, and the pellet was
resuspended in 10 ml of deionized water and washed using the
tube mixer. The washing-centrifugation process was repeated ve
times to remove the residual alkali. The washed pellet was then
mixed with 10 ml of deionized water and neutralized with an
appropriate amount of 1 M hydrochloric acid to a pH of 4.8, the
mixture again centrifuged, and the supernate discarded. The pellet
was nally washed with 10 ml of deionized water or 50 mM sodium citrate buffer (pH 4.8) for compositional analysis or enzymatic hydrolysis experiment.
2.2.2. Dilute sulfuric acid pretreatment
Ground bagasse samples (each 140 mg) were mixed with 2.8 ml
of 0.5% (w/w) sulfuric acid in glass tubes. The tubes were t into
custom-made stainless steel mini-reactors, and placed in an oil
bath at 170 C for different time periods. After treatment, the reactors were immediately quenched in ice bath to terminate the reactions. Pretreatment liquids were collected by centrifugation
(12,000g, 5 min), and neutralized with calcium carbonate. Sugar
monomers including glucose and xylose present in the liquids
were quantied using an HPLC system (Prominence UFLC, Shimadzu, Japan) equipped with a refractive index detector (RID-10A,
Shimadzu, Japan) and carbohydrate-analysis column (Aminex
HPX-87P, Bio-rad, USA). The pellets were washed with deionized
water until pH > 5 for compositional analysis and enzymatic
hydrolysis experiment.

Table 1
Low temperature alkali and dilute sulfuric acid pretreatment conditions.
Pretreatment

Parameter

Scope

LTA

NaOH concentration (mol/l)

Solid to liquid ratio (%, w/v)
Temperature (C)
Treatment time (h)

0.55
5, 10, 15
25, 50
0.524

Dilute sulfuric acid

Concentration (%, w/w)

Solid to liquid ratio (%, w/v)
Temperature (C)
Treatment time (min)

0.5
5
170
560

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L. Wu et al. / Bioresource Technology 102 (2011) 47934799

Detailed experimental pretreatment conditions are listed in

Table 1.

Table 2
Major components of sweet sorghum bagasse.
Component

2.3. Compositional analysis

After pH adjustment and washing, the alkali and acid pretreated
solids were dried in a drying oven at 65 C to constant weight and
recomminuted using the blender. The composition (including
moisture, soluble sugars, glucan, xylan, lignin, ash content, etc.)
of the pretreated as well as untreated bagasse was analyzed following modied NREL laboratory analytical procedures (NREL).
According to the NREL LAP, a two-step sulfuric acid hydrolysis process was adopted to break down the structural polysaccharides
into sugar monomers for quantication (by the HPLC system);
the acidinsoluble lignin (AIL) present in the acid hydrolysis residue was then determined gravimetrically.
2.4. Enzymatic hydrolysis (saccharication)
One milliliter of preheated (50 C) enzyme solution, comprising
commercially available cellulase and b-glucosidase (Celluclast 1.5L
and Novozyme 188 (Novozymes A/S, Denmark) in 3:1 ratio in
50 mM pH 4.8 sodium citrate buffer), was added to a test tube containing alkali or acid pretreated bagasse (pellet) or 140 mg of untreated bagasse (approximate enzyme loading: 20 FPU, 50 CbU/g
glucan). The mixture was immediately incubated in a shaking
water bath at 50 C for a certain period of time (ranging from 1
to 96 h). The hydrolysis process was terminated by heating the
tube in a boiling water bath for 15 min. After cooling down to room
temperature, the hydrolysate was analyzed with the HPLC system
to determine the released sugars. The hydrolysis performance of
the cellulose in the biomass was evaluated by saccharication
yield dened as follows:

Glucan
Xylan
AIL
Ash
a

Content (%, d.b.)a

Kyushuko4

SIL 05

38.7 0.4
22.6 0.8
15.4 0.7
2.2 0.2

37.8 1.1
21.2 0.3
16.7 0.6
2.4 0.4

Mean SE; n P 5.

fructose, etc.) present in the juice were substantially removed from

the bagasse, avoiding possible interference in the evaluation of the
hydrolysis efciency of the cellulose. According to the two-step
sulfuric acid hydrolysis method for the determination of the structural components of lignocellulose, the bagasse of Kyushuko4 had
slightly higher glucan and xylan contents but somewhat lower
AIL content compared with the SIL05 bagasse, as shown in Table 2.
Additionally, the ash contents of the Kyushuko4 and SIL05 bagasse
would be noteworthy as the ash (especially silicates) in the feedstocks may affect the efciency of pretreatment chemical recycle
process.
The LTA pretreatment drastically changed the composition of
the bagasse. Fig. 1(a) and (b) shows the recovery rates of the glucan
and xylan contained in the Kyushuko4 and SIL05 bagasse after pretreatment with NaOH solutions of different concentrations (0.5
2.5 M for Kyushuko4; 15 M for SIL05) at room temperature for
30120 min. The pretreatment caused only minor glucan loss,
and the loss rates were unaffected by pretreatment condition;

Glucan saccharification yield %

100 

0:9  released glucose after hydrolysis

initial glucan before hydrolysis

The enzymatic hydrolysis of the dilute sulfuric acid treated bagasse was performed likewise. Some other auxiliary experiments
were also detailed in Section 3. All pretreatment-saccharication
experiments were carried out in duplicate. Data are presented as
mean SE. Comparisons between means were performed using
Duncans multiple range tests at a signicance level of 0.05.
2.5. X-ray powder diffraction analysis
Samples of the untreated ground bagasse and freeze dried LTA
pretreated bagasse were pressed (200 kgf/cm2, 30 s) into disks
for X-ray powder diffraction analysis. The diffractometry in reection mode was carried out using an X-ray diffractometer
(RINT2000, Rigaku, Japan) with monochromatic Cu Ka radiation
(k = 0.15418 nm) generated at 38 kV and 50 mA, and the following
optical slit system: divergence slit = 0.5; scattering slit = 0.5; and
receiving slit = 0.15 mm. The scanning was performed at scattering
angles (2h) from 6 to 30 in 0.1 increments with an accumulation
time of 20 s for each angular step.
3. Results and discussion
3.1. Effects of LTA treatment on the composition and structure of
bagasse
After juice extraction, washing and drying processes, about
180 g of dry bagasse was recovered from 1 kg of each variety of
sweet sorghum stems. The major soluble sugars (sucrose, glucose,

Fig. 1. Effects of LTA pretreatment under different conditions on the glucan and
xylan in sweet sorghum bagasse.

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L. Wu et al. / Bioresource Technology 102 (2011) 47934799

about 95% of the glucan present in the untreated bagasse was

recovered after pretreatment. By contrast, a large portion of xylan
was degraded and removed during the pretreatment, and the removal rate increased rapidly with increasing pretreatment severity. For instance, 1-h LTA treatment with 1 M NaOH led to about
33% and 54% loss of the xylan of the SIL05 and Kyushuko4 bagasse,
respectively, while the loss rates increased to 62% and 75% when
the bagasse was treated with 2.5 M NaOH.
Fig. 2 presents residual acid-insoluble lignin in the LTA pretreated bagasse (as percentage of initial AIL amount). The lignin
content of the biomass was signicantly reduced after the pretreatment at moderate temperatures. In Fig. 2a, only 45% of the initial
AIL remained in the Kyushuko4 bagasse after 30 min 0.5 M NaOH
treatment at room temperature. The AIL removal rate increased
slowly with extended pretreatment to about 67% at 120 min. The
delignication reaction accelerated markedly with increasing
NaOH concentration. Only about 20% of the initial AIL remained
in the Kyushuko4 bagasse after 2.5 M NaOH treatment for
120 min. Similar trend was also observed in the SIL05 bagasse
(Fig. 2b), although more severe treatment conditions were needed
to achieve an equal level of delignication in comparison to the
Kyushuko4 bagasse. In biomass pulping, the course of delignication shows three stages: initial, bulk, and residual stage. The
three-stage model has been widely used to study the delignication kinetics of the kraft/soda pulping (Chiang et al., 1990; Kim
and Holtzapple, 2006b). Compared with the data in the reports discussing the delignication kinetics, the delignication of the bagasse under the present experimental conditions occurred
rapidly in the initial and bulk phases, and then slowed down significantly as the pretreatment proceeded.
Temperature may strongly affect the rates of chemical reactions
and heat and mass transfer as well. It was observed in this study

Fig. 2. Residual acid-insoluble lignin in LTA pretreated sweet sorghum bagasse.

that the higher treatment temperature (50 C) signicantly accelerated the removal of hemicellulose and lignin. The delignication
rate could reach as high as 90% while the glucan recovery rates remained almost unaffected by the elevated temperature (data not
shown).
The data in Figs. 1 and 2 also exhibited that the Kyushuko4 bagasse was more susceptible to the pretreatment compared with
the SIL05 bagasse. The former achieved higher xylan and lignin removal rates under the same pretreatment conditions. In other
words, in order to achieve similar compositional changes (glucan,
xylan and AIL content), more severe treatment conditions (e.g.
higher alkali concentration, treatment temperature or longer treatment time) would be needed for the SIL05 bagasse.
Alkali treatment of natural crystalline cellulose (such as the
mercerization process in the textile industry) can change the crystal structure and thus alter its physical and chemical properties
(Nishiyama et al., 2000; Oh et al., 2005). The effect of the LTA pretreatment on the cellulose crystal structure of the bagasse was
investigated by means of X-ray powder diffractometry. Fig. 3 compares the X-ray diffraction proles (diffraction intensity against
diffraction angle 2h) of the untreated and the bagasse treated with
1, 2.5 and 5 M NaOH at room temperature for 60 min. Overall, the
diffractograms of the Kyushuko4 and SIL05 bagasse samples
showed little difference, and the diffraction patterns of the untreated samples were similar to those of 1 or 2.5 M NaOH treated
samples, where the diffraction peaks at 2h of 14.6, 16.1 and
 and 002 lattice planes of the cellulose
21.8 indicating the 101, 101
I crystal structure of higher plants (Parikh et al., 2007) were clearly
identiable. In contrast, the 5 M NaOH treated samples demonstrated a distinctly different pattern, in which the diffraction intensities at 14.6 and 16.1 substantially decreased, while a new peak
at 12.1 stood out; the shape of the major peak at 21.8 also changed drastically. The new pattern, when compared with those of the

Fig. 3. X-ray diffractograms of untreated and LTA pretreated sweet sorghum

bagasse samples.

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4797

mercerized or regenerated cellulose, basically agreed with the

crystal lattice characteristics of cellulose II crystal structure, suggesting that a certain amount of crystalline cellulose could transform from cellulose I to cellulose II during the pretreatment; the
treatment conditions under which apparent crystal structure
changes occurred were comparable to those reported in the literature (Isogai, 1998; Mansikkamki et al., 2005). It is generally believed that cellulose I has parallel glucan chains arranged
uniaxially, whereas glucan chains of cellulose II are arranged in a
random manner (mostly antiparallel) and linked with a larger
number of hydrogen bonds resulting in higher thermodynamic stability of the structure (Kolpak and Blackwell, 1976). Other reports
(Kolpak et al., 1978; Mannan, 1993) also pointed out that the crystal structure transformation of lignocellulosic biomass was not as
complete as that of relatively clean cellulose such as cotton linter
or bacterial cellulose; the discrepancy was mainly attributed to the
lignin and hemicellulose surrounding the cellulose brils in the lignocellulose, for their prevention of the swelling of the cellulose
which was considered the starting point of the crystal structure
transformation process.
The crystallinity of the LTA pretreated bagasse was evaluated by
crystallinity index (CrI), which has been extensively used to estimate the ratio of cellulose I crystal structure to amorphous region
in a biomass sample. The CrI values were calculated according to
the following equation using the data from the diffractograms
(Fig. 3),

CrI % 100  I002  Iamorphous =I002 

where I002 is the intensity of diffraction peak at 2h of 21.8;
Iamorphous is the intensity attributed to amorphous portion at 2h
of 18 (Segal et al., 1959).
As presented in Table 3, the indexes of the untreated bagasse
samples were about 50% due to the existence of a large amount
of amorphous substances including lignin and hemicellulose. After
1 M NaOH treatment, the CrI increased by about 10%, suggesting
there was a remarkable increase in the relative amount of crystalline matter with the removal of the amorphous components. The
CrI showed a tendency to decline with increasing NaOH concentration, which could be attributed to the crystal structure change, cellulose amorphization or the changes in the microscopic structures
of the lignocellulose particles after the pretreatment.
3.2. Effect of LTA pretreatment on the enzymatic digestibility of
bagasse
Without any pretreatment, only about 29% and 26% of the cellulose present in the Kyushuko4 and SIL05 bagasse was hydrolyzed
into glucose after 24 h hydrolysis under the present hydrolysis
conditions. Fig. 4 shows the 24-h saccharication yield of glucan
from the bagasse pretreated with 0.5 to 5 M NaOH at room temperature for 30 to 120 min. The enzymatic digestibility of the biomass
was greatly improved after the pretreatment. In the case of Kyushuko4 (Fig. 4a), for example, the saccharication yield of the bagasse
treated with 0.5 M NaOH for 30 min reached 80%. The digestibility
of the pretreated bagasse was positively correlated with pretreat-

Table 3
Crystallinity index of untreated and LTA pretreated sweet sorghum bagasse samples.
Treatment

Untreated
1 M NaOH
2.5 M NaOH
5 M NaOH

CrI (%)
Kyushuko4

SIL 05

51.8
61.3
54.3
42.8

50.8
57.5
48.0
38.8

Fig. 4. Twenty-four-hour glucan saccharication yield of LTA pretreated sweet

sorghum bagasse.

ment severity, especially with the concentration of NaOH solution.

More than 90% of the cellulose could be hydrolyzed into glucose
within 24 h when 1 M or above NaOH solution was used. A maximum 24-h saccharication yield of 98.7% was observed when the
bagasse was pretreated with 2.5 M NaOH at room temperature
for 120 min. The pretreated SIL05 bagasse also showed markedly
improved enzymatic digestibility after pretreatment; the 24-h saccharication yield increased from 65% to 90% as the pretreatment
intensied (from 1 M NaOH 30 min to 5 M NaOH 120 min), as
shown in Fig. 4b. Compared with the Kyushuko4 bagasse, the
SIL05 bagasse treated under the same conditions had apparently
lower digestibility. It achieved comparable saccharication yield
only when treated under more severe conditions, which was in
accordance with its susceptibility to the pretreatment. Considering
the higher digestibility of the untreated Kyushuko4 (BMR mutant)
bagasse, the difference in the susceptibility and enzymatic digestibility between the two varieties could be due to their different
structural features, since there were only slight differences in their
composition. Therefore, using the BMR sweet sorghum bagasse as
feedstock for ethanol production could contribute to achieving
high biomass-to-ethanol conversion efciency.
In addition, the xylan remained in the pretreated bagasse was
also degraded by the commercial cellulases during the saccharication processes; about 6070% of the residual xylan in the above
pretreated Kyushuko4 and SIL05 bagasse samples was hydrolyzed
into xylose after 24-h hydrolysis. The hydrolysis rate of the xylan
increased more moderately with increasing pretreatment severity
in comparison to the glucan. The nal xylose concentration of
the hydrolysate depended largely on the amount of xylan survived
the pretreatment.
The enzymatic digestibility of the pretreated biomass improved
with increasing pretreatment severity in this study. Nevertheless,
the practical pretreatment conditions must be chosen comprehen-

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L. Wu et al. / Bioresource Technology 102 (2011) 47934799

sively, taking various factors into consideration in addition to the

saccharication yield, such as feedstock characteristics, pretreatment chemical cost, recovery efciency and energy consumption.
3.3. Mechanisms of improvement in the enzymatic digestibility of
bagasse
The proposed pretreatment method effectively improved the
enzymatic digestibility of the lignocellulosic biomass. Additional
experiments were carried out to clarify the relationship between
the compositional and structural changes and the enzymatic
digestibility improvement of the pretreated bagasse.
Firstly, 10 g of each kind of bagasse were treated with 2.5 M
NaOH at 50 C for 24 h to remove the majority of the lignin. After
washing, neutralizing, drying and recomminution, delignied bagasse samples, each containing 50 mg glucan, were again treated
with 15 M NaOH or citrate buffer (control) at room temperature
for 2 h. The samples (pH adjusted) were then hydrolyzed under
the same enzymatic hydrolysis conditions as described in Section 2.4 except that 0.5-fold diluted enzyme solution (half enzyme
loading) was used so as to slow down the enzyme reactions and
magnify the characteristics of the substrate. The two-step alkali
treatment still caused slight glucan loss (<5%); the rst step treatment removed about 87% and 81% of the acid-insoluble lignin from
the Kyushuko4 and SIL05 bagasse respectively, and the second step
treatment slightly increased the removal rates to about 90% and
85% due to the signicantly reduced reaction rate in the late bulk
delignication stage. There was no signicant difference in the
residual lignin content between the samples treated with different
NaOH solutions in the second step. The enzymatic hydrolysis
experiment showed that the samples treated with different NaOH
solutions had quite similar hydrolysis performance, with little differences in both initial (1 h) hydrolysis rate (Kyushuko4: about
25%; SIL05: about 22%) and 24-h saccharication yield (about
83% and 78% from Kyushuko4 and SIL05, respectively), indicating
that the differently treated samples having similar composition
were equally digestible. The control groups treated with buffer in
the second step had slightly lower digestibility than the alkali treated samples, giving 24-h glucose yields of about 78% (Kyushuko4)
and 70% (SIL05). In association with the results of X-ray diffraction
analysis, the effect of the changes in the cellulose crystal structure
or crystallinity caused by the pretreatment on the enzymatic
digestibility of the pretreated bagasse was insignicant. The difference in crystallinity between the differently treated samples could
hardly be attributed to the amorphization of the cellulose either;
otherwise, their hydrolysis proles would be distinct from one another since amorphous structure is much more susceptible to
enzymatic hydrolysis. Accordingly, the digestibility improvement
after the pretreatment can be largely attributed to the disruption
of the protective hemicelluloselignin matrix surrounding the cellulose microbrils by the pretreatment, which caused increase in
the accessibility of the cellulose to enzymes.
The two-step pretreated Kyushuko4 also showed higher enzymatic digestibility than the SIL05 bagasse, suggesting the existence
of the structural difference between the two varieties. Moreover,
the saccharication yield from both varieties reached about 80%
at 24 h even at half enzyme loading, and kept increasing to up to
95% at 72 h, indicating a great potential of the LTA pretreatment
method for the reduction of enzyme dosage for the saccharication
of the biomass.
Dilute sulfuric acid pretreatment of the bagasse was also performed for comparison. The pretreatment conditions (0.5% H2SO4
at 170 C) were selected according to preliminary optimization
experiments, and basically consistent with the conditions reported
elsewhere (Cara et al., 2008; Saha et al., 2005; Torget et al., 1990).
The xylose present in the acid pretreatment supernates (as per-

centage of initial xylan amount) of the Kyushuko4 and SIL05 bagasse samples treated for different time periods, and the 24-h
glucan saccharication yield of corresponding pretreated solids
are given in Fig. 5. The xylan degraded rapidly into xylose during
the acid pretreatment. The amount of xylose in the pretreatment
liquids reached the maximum, equaling about 85% of the xylan
present in the untreated bagasse, after 20-min treatment. Excessive treatment then caused gradual degradation of the xylose,
resulting in a decrease in the xylose concentration of the liquids.
The saccharication yield of the acid pretreated bagasse increased
at a steady rate to about 65% until the maximum xylan removal
rate was reached; acid pretreatment over 20 min showed little
additional effect on the improvement in the enzymatic digestibility
of the bagasse, which never exceeded 70% in this study. In contrast,
The LTA pretreatment resulted in greater digestibility improvement while a certain amount of xylan remained in the pretreated
bagasse. For example, the Kyushuko4 and SIL05 bagasse treated
with 1 M NaOH for 120 min achieved 24-h saccharication yield
of 90% and 75%, respectively, with about 40% and 60% of the initial
xylan (before pretreatment) present in the pretreated solids (Figs. 1
and 4). Because dilute sulfuric acid pretreatment has minor impact
on the amount of acid-insoluble lignin and the crystal structure of
cellulose (Kabel et al., 2007; Kumar et al., 2009), the improvement
in the digestibility of the acid pretreated bagasse over the untreated feedstock can be largely attributed to the increase in cellulose accessibility due to the removal of the hemicellulose.
Therefore, it can be concluded that the hemicellulose made a relatively small contribution to the recalcitrance to hydrolysis, while
the lignin decisively affected the enzymatic digestibility of the cellulose. The removal of lignin brought signicant increase in the
available surface area of the cellulose and reduced nonproductive
binding of enzymes, and, consequently, led to the great enhancement in the accessibility of the substrate to enzymes and resulting
cellulose hydrolysis efciency. Sulfuric acid pretreatment attacking
mainly the hemicellulose fraction of lignocellulosic biomass was
inferior to the LTA pretreatment in improving the digestibility of
the biomass.
The 24 h saccharication yield (full enzyme loading) of LTA pretreated Kyushuko4 and SIL05 bagasse as a function of lignin removal rate is plotted in Fig. 6, regardless of specic treatment
conditions (NaOH concentration, treatment time, etc.). There was
an apparently positive, linear correlation between the two
variables; the efciency of enzymatic hydrolysis markedly increased as the lignin content of the bagasse dropped. Adsul et al.
(2005) obtained similar results using differently delignied sugar-

Fig. 5. Effect of dilute sulfuric acid pretreatment on 24-h glucan saccharication

yield of sweet sorghum bagasse.

L. Wu et al. / Bioresource Technology 102 (2011) 47934799

Fig. 6. Correlation between lignin removal and enzymatic digestibility of LTA

pretreated sweet sorghum bagasse.

cane bagasse as substrate under comparable enzymatic hydrolysis

conditions. The correlation coefcient for the Kyushuko4 bagasse
was lower than that for the SIL05, owing to the uneven distribution
of the experimental data of the former (mostly very high delignication rates and sharply increased enzymatic digestibility).
Thus, the LTA pretreatment, derived from the highly mature
kraft/soda pulping technology focusing on removing lignin from
lignocellulosic feedstocks, may act as an effective means for
improving the enzymatic digestibility of lignocellulosic biomass.
Additionally, due to the substantially reduced lignin content of
the LTA treated biomass, the downstream unit operations such as
distillation and waste treatment may also be facilitated. The present method can be integrated with other advanced biorenery
technologies to build cost-effective second generation bioethanol
production platforms.
4. Conclusions
The LTA pretreatment method proposed in this study can effectively disrupt the lignincarbohydrate complex and liberate the
cellulose brils in the sweet sorghum bagasse by removing the
majority of the lignin, and therefore greatly enhance the enzymatic
digestibility of the cellulose. The bagasse from the BMR mutant
sweet sorghum is more susceptible to the pretreatment than the
non-BMR variety, leading to a higher enzymatic hydrolysis efciency of the former, which may also provide useful information
for the development of novel feedstocks for cellulosic ethanol production. The method offers an alternative approach to the efcient
conversion of lignocellulosic biomass to ethanol.
Acknowledgement
This work was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Rural Biomass Research
Project, BEC-BA230).
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